The present invention relates to a method and apparatus for automating nucleic acid sample preparation.
Sequencing of nucleic acids is generally performed today using the chain termination sequencing method first described by Sanger et al. This process generally involves combining a sample to be analyzed with reagents necessary for synthesis of nucleotide fragments indicative of the sequence of DNA in the sample, i.e, a polymerase enzyme, nucleoside feedstocks and a dideoxynucleoside for chain termination, followed by analysis of the fragments produced by gel electrophoresis. In addition, the sample may be initially amplified, i.e., by polymerase chain reaction (PCR) amplification, to increase the amount of a selected part of the DNA in an original sample prior to performing the sequencing reaction. Thermally stable polymerases, such as Taq Polymerase (Hoffman-La Roche, Inc.) and improved thermal stable chain-termination sequencing enzymes such as Thermo-Sequenase.TM. (Amersham Life Science, Cleveland) are used to enhance these procedures. These enzymes are utilized in conjunction with multiple successive thermal cycles for amplification or sequencing.
The use of DNA diagnostics in clinical laboratories is not widespread, in part because of the complex and cumbersome steps required to prepare DNA samples for analysis. To overcome this problem, it would be advantageous to have an apparatus which automated the large number of pipetting steps and the thermocycling steps involved in preparing a DNA sample for sequence analysis. At the present time, however, most known robotics have handled either the task of pipetting reagents and sample material to produce a reaction mixture or the task of thermal cycling of the reaction mixture, but not both.
For example, Beckman Instruments, Inc. (Fullerton, Calif.) provides the Biomek.RTM. 2000 automated pipetting apparatus, that can automate the sample preparation steps for PCR or DNA sequencing reactions in a 96 well microtiter plate using a group of eight pipetting tips. Trays containing reagents or samples are arranged for sequential liquid transfer functions. Another pipette robot, the QIAGEN BioRobot.TM. 9600 (QIAGEN Inc., Chatsworth Calif.) can prepare 96 bacterial minipreps in 2 hours. These robots all use a cooling plate to keep the reagents and samples at controlled temperatures (usually 4 degrees C.) during sample preparation.
The reagent trays prepared in an apparatus of this type are then generally transferred to a separate instrument for purposes of thermal cycling. For example, the RoboCycler.TM. Gradient 96 System (Stratagene, Inc.) has 4 different temperature blocks and a lifter that moves a tray of up to 96 tubes from block to block in sequence. In this way, the apparatus cycles reaction mixtures through a series of temperature increases and decreases as appropriate for amplification or sequencing reactions.
Heating of the temperature blocks in thermal cycling apparatus has been accomplished in a variety of ways, including heating and cooling using liquid flow or electronic means (e.g., resistive heating). An air flow thermal cycling mechanism is made by Idaho Technology (Idaho Falls, Id.). To use this apparatus, capillary tubes containing the desired reaction mixtures are melt-sealed at both ends with a Bunsen burner. The permanently sealed capillaries are inserted through and held in a rubber plug. The plug is placed into an opening in a heat chamber, such that the sealed capillaries are suspended in the heat chamber without touching the walls of the chamber. Air heated to the desired temperature is circulated through the heat chamber, thereby heating and cooling the capillaries as programmed. Upon completion of the desired thermal cycles, the capillaries may be removed from the rubber plug. The capillaries are then broken at both ends, and the reaction mixture is extracted for analysis or for mixing with further reagents. Drawbacks of using this apparatus include that the handling of the capillaries is time consuming and that the procedure requires the use of multiple capillaries for each treatment of the same sample.
While there are many potential advantages to combining sample preparation and thermal cycling into a single apparatus, only one apparatus known to Applicants actually attempts such a combination. The Vistra DNA Labstation 625 (Molecular Dynamics, Sunnyvale Calif.) is a pipette robot that can prepare bacterial mini-preps and PCR and DNA sequencing reactions in a 96 well microtiter plate. The Labstation 625 has an integrated Peltier-block thermocycler for thermal-cycling steps. Using this apparatus, a technician can prepare a sequencing experiment in about 10-15 minutes, and then start the thermocycling procedure. This apparatus uses tubes, and places a layer of oil on top of the reactions to reduce loss of sample during heating. One of the drawbacks of this device, is the long time required for temperature changes due to the use of a heating block.
It is an object of the present invention to provide an improved thermocycler, which can be combined with a sample preparation apparatus if desired.
It is another object of the present invention to provide an apparatus and method for automating the preparation of nucleic acid samples for rapid throughput.
It is a further object to provide a method and apparatus that integrate automation of pipetting functions with thermal cycling to eliminate manual handling and to optimize reaction conditions.
It is further object of the invention to provide a method and apparatus that minimize the risk of contamination between samples and that permit sterility to be maintained throughout the procedure.
It is a further object of the invention to provide an apparatus which is capable of loading prepared nucleic acid samples into an automated DNA sequencing apparatus or onto an electrophoretic analytical gel.